Exosome for synergistically inhibiting copper death based on double pathways as well as preparation method and application of exosome

Abstract

The invention discloses an exosome for synergistically inhibiting copper death based on double pathways and a preparation method and application thereof, and relates to the technical field of biological medicine, the main points of the technical scheme are that the exosome is a polydopamine modified stem cell exosome, the polydopamine modified stem cell exosome is loaded with a copper chelating agent and siRNA-FDX1, the siRNA-FDX1 is used for inhibiting the expression of the ferredoxin 1. According to the stem cell exosome modified by the Cu-chelating agent polydopamine containing the siRNA-FDX1, two ways of effectively inhibiting copper death can be synthesized, and the functionalized exosome is loaded into injectable gel and can be locally and controllably released, so that the retention of a therapeutic agent on a wound surface is ensured, the wound surface healing is effectively promoted, and the curative effect is good. Meanwhile, the engineering exosome strategy with the functional nanoparticles, provided by the invention, provides an effective and extensible novel method for adjusting copper death.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to exosomes that synergistically inhibit copper death based on dual pathways, and a preparation method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) are key to controlling various processes in typical wound healing, such as inflammation, cell growth, and intracellular metal ion levels. After injury or in a long-term refractory wound bed, ROS levels rise sharply, causing cells to suffer excessive oxidative stress damage, thereby overactivating superoxide dismutase (SOD) and uncontrollably increasing the copper (Cu) content in cells; therefore, excessive copper increases can lead to an imbalance in intracellular copper homeostasis, resulting in oocyte apoptosis. Luteogenesis is mainly regulated by two pathways:

[0003] (I) Intracellular Cu 2+ The overload is reduced to the more toxic Cu + , thereby destroying the stability of Fe-S cluster proteins and leading to cell death;

[0004] (ii) The key regulatory molecule ferritin 1 gene (ferredoxin 1 (FDX1)) affects the mitochondrial tricarboxylic acid cycle and causes cell death. However, copper death has been reported in various diseases, but there is a lack of experimental exploration of chronic non-infectious wounds.

[0005] Exosomes have recently been widely used as gene carriers due to their unique lipid bilayer structure, excellent biocompatibility and easy accessibility. These vesicles have shown promising therapeutic effects in many animal disease models and clinical studies. However, due to the different loading efficiency and molecular stability of miRNA, siRNA and drugs in liposomes, liposomes are currently mainly used as single substance delivery carriers; Currently, the modification of catechol nanodrugs has become one of the most effective methods for upgrading exosomes. Exosomes are widely used to cover various surfaces, such as bacteria and cells, to achieve controllable drug delivery. Exosomes are usually applied to the wound bed using the traditional peri-wound injection method, which can lead to uneven drug distribution and rapid loss of drugs within a few hours, thus affecting the therapeutic effect. At the same time, this often requires repeated injections, causing inconvenience and additional treatment costs. When exosomes are encapsulated in injectable hydrogels, uniform drug distribution can be achieved on the wound bed, while reducing drug dosage and promoting slow and controlled release.

[0006] Therefore, the present invention aims to provide exosomes based on dual-pathway synergistic inhibition of copper death and a preparation method and application thereof, so as to solve the above problems. Summary of the invention

[0007] The purpose of the present invention is to provide exosomes based on dual-pathway synergistic inhibition of copper death, and a preparation method and application thereof. The present invention synthesizes a Cu-chelator polydopamine-modified stem cell exosome containing siRNA-FDX1, which can effectively inhibit the two pathways of copper death. The functionalized exosomes are loaded into an injectable gel, which can be locally and controllably released, ensuring the retention of the therapeutic agent on the wound bed, effectively promoting wound healing. At the same time, the engineered exosome strategy with functional nanoparticles proposed in the present invention provides an effective and scalable new method for regulating copper death.

[0008] The above technical purpose of the present invention is achieved through the following technical scheme: an exosome based on dual-pathway synergistic inhibition of copper death, wherein the exosome is a polydopamine-modified stem cell exosome, and the polydopamine-modified stem cell exosome is loaded with a copper chelator and siRNA-FDX1, and the siRNA-FDX1 is used to inhibit the expression of ferredoxin 1.

[0009] The present invention also provides a method for preparing exosomes based on dual-pathway synergistic inhibition of copper death, comprising the following steps:

[0010] S1, extracting exosomes from bone marrow mesenchymal stem cells using gradient centrifugation;

[0011] S2, loading siRNA-FDX1 into exosomes using water bath sonication technology;

[0012] S3, preparing polydopamine nanoparticles by a self-polymerization method, and loading a copper chelator to form a nanodrug of polydopamine loaded with a copper chelator;

[0013] S4. The nanodrugs loaded with copper chelators such as polydopamine were combined with the surface of exosomes through nano-contact vortex technology to complete the preparation of exosomes based on dual-pathway synergistic inhibition of copper death.

[0014] The present invention also provides an application of exosomes based on dual-pathway synergistic inhibition of copper death in promoting wound healing.

[0015] The present invention also provides an application of exosomes based on dual-pathway synergistic inhibition of copper death in the preparation of a drug for treating wound healing.

[0016] The present invention also provides a pharmaceutical composition, comprising the exosomes based on dual-pathway synergistic inhibition of copper death and a hydrogel carrier as described in claim 1, wherein the hydrogel carrier encapsulates the exosomes based on dual-pathway synergistic inhibition of copper death, and is used to prolong the retention time of the exosomes based on dual-pathway synergistic inhibition of copper death.

[0017] In summary, the present invention has the following beneficial effects:

[0018] 1. The engineered exosomes of the present invention achieve dual-pathway synergistic inhibition of copper death by combining copper ion chelation and siRNA-FDX1 gene silencing technology. This innovative dual-pathway strategy can not only immediately chelate excess copper ions in cells and reduce the occurrence of copper death, but also downregulate the expression of FDX1 for a long time, thereby inhibiting copper death at the molecular level and improving the durability and stability of the therapeutic effect;

[0019] 2. The present invention has verified in animal models that the engineered exosomes of the present invention can effectively promote the healing of diabetic wounds and acute skin wounds. This promotion effect is due to the dual-pathway synergistic inhibition of exosomes, which reduces cell death caused by copper death and provides a more favorable microenvironment for wound healing;

[0020] 3. The engineered exosomes of the present invention utilize the natural advantages of exosomes, such as excellent biocompatibility and targeting, to improve the efficiency and accuracy of drug delivery. In particular, through the modification of PDA@CQ nanomedicines, the stability and drug encapsulation capacity of exosomes are enhanced, so that the therapeutic agent can reach the lesion site more effectively.

[0021] 4. By loading engineered exosomes into injectable hydrogels, the present invention achieves local and controlled release of drugs on the wound bed. This sustained-release system not only improves the utilization efficiency of drugs, but also reduces toxic side effects on normal tissues, reduces treatment costs and patient suffering;

[0022] 5. The materials used in the present invention, including exosomes, PDA@CQ nanomedicines and hydrogels, have good biocompatibility, reduce immune responses and side effects during treatment, and improve the safety of treatment;

[0023] 6. The engineered exosomes of the present invention and their preparation method and application provide a new treatment strategy for the management of clinically refractory wounds, especially in the treatment of diabetic wounds and acute skin wounds. The present invention shows great potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the workflow of the therapeutic application of engineered exosomes modified with nanomedicine in an embodiment of the present invention;

[0025] Figure 2 This is a result diagram of copper death hindering diabetic wound healing in an embodiment of the present invention;

[0026] Figure 3 Schematic diagram of constructing engineered exosomes modified with nanomedicine in an embodiment of the present invention;

[0027] Figure 4Schematic diagram of preparation and evaluation of hydrogel in an embodiment of the present invention;

[0028] Figure 5 EXO in the embodiment of the present invention siFDX1-PDA@CQ Pathways synergistically inhibit copper death results in time and space;

[0029] Figure 6 EXO in the embodiment of the present invention siFDX1-PDA@CQ Results of effectively promoting diabetic wound healing;

[0030] Figure 7 EXO in the embodiment of the present invention siFDX1-PDA@CQ The results of accelerating the healing of diabetic wounds by reducing Cu content and inhibiting the expression of FDX1;

[0031] Figure 8 This is a diagram showing the functional verification results of the engineered exosomes modified with nanomedicine in the full-thickness pig skin wound model in an embodiment of the present invention. DETAILED DESCRIPTION

[0032] The following is combined with Figure 1-8 The present invention is described in further detail.

[0033] The cells and culture conditions in this example are as follows: NIH-3T3 and HUVECs (Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in high glucose Dulbecco's modified Eagle medium (DMEM, Pricella) supplemented with 10% fetal bovine serum (FBS, Pricella) and 1% penicillin and streptomycin (Gibco). NIH-3T3 and HUVECs were used for cell line identification. All cell lines were cultured in a humidified incubator at 37°C and 5% CO2, and cells were studied in vitro using various concentrations of engineered exosomes.

[0034] Source of wound tissue and detection of tissue copper and mRNA levels: Tissues were obtained from the wound sites of type 1 diabetic mice, and wounds of normal and diabetic mice were collected for follow-up examination within three days. RNA-seq analysis (Novogene, China) was performed on the relevant tissues to evaluate Cu levels. The Cu microplate kit was purchased from Absin Bioscience (Shanghai, China), and the liquid samples were wound tissue lysates extracted from 20 healthy mice or type 1 diabetic mice. The experiments were performed according to the manufacturer's instructions.

[0035] Immunofluorescence analysis: To evaluate the expression of FDX1 in healthy human and type 1 diabetic wound tissues, mouse skin samples were fixed with 4% glutaraldehyde for 3 days and then embedded in paraffin and sectioned. The samples were incubated with the primary antibody against FDX1 (Abcam, USA) overnight at 4°C. The next day, the sections were incubated with the secondary antibody (Boster, China) at room temperature and then stained with DAPI (Yeasen, China) for nuclear staining and mounted. Images were captured using a fluorescence microscope. Three different samples were evaluated in each group. Finally, ImageJ software (NIH, ImageJ1.8, USA) was used for analysis.

[0036] Western blotting: Tissue lysates were extracted with RIPA lysis buffer (Beyotime, China) containing 1% PMSF solution (Beyotime, China) and 1% protease inhibitor (Beyotime, China) at 4°C for 20 minutes, and equal amounts of total protein (20-50 μg) were separated by SDS-PAGE (Epizyme, China) and then separated using FDX1 (Abcam, USA), nicotinamide adenine dinucleotide (Abcam, USA) and β-actin (Proteintech, China). The membrane was incubated with the secondary antibody (Boster, China), then exposed to X-ray film (UVP, USA), and analyzed by blotting using ImageJ software (NIH, ImageJ1.8, USA).

[0037] High-throughput sequencing: The analysis was performed using Novogene software (Beijing, China). Briefly, wound tissues were collected from type I diabetic mice, healthy mice, and normal skin, immediately frozen in liquid nitrogen, and stored at −80°C, then, mRNA was purified from total RNA using magnetic beads bound to poly(T) oligomers, and library fragments were purified using the AMPure XP system to preferentially select cDNA fragments with a length of 370 to 420 bp, then, qRT-PCR was performed using Phusion high-fidelity DNA polymerase, universal qRT-PCR primers, and Index(X) primers, and RNA integrity was assessed using the RNANano 6000 assay kit of the Bioanalyzer 2100 system (Agilent Technologies, California, USA). Index-encoded samples were clustered on the Bot Cluster Generation System using the TruSeq PE Cluster Kit v3 cBot HS (Illumina) according to the manufacturer’s instructions, and after cluster generation, libraries were sequenced using the Illumina NovaSeq platform to generate 150 bp paired-end reads.

[0038] RNA sequencing and bioinformatics analysis: Bioinformatics analysis of RNA-seq data was performed using R software (version 4.0.3), and the “FactoMineR” and “factoextra” packages were used for PCA. DEGs were analyzed using the “Limma” package (FDR < 0.05, |log FC| > 2). Enrichment analysis was performed using the “ClusterProfiler” package.

[0039] Isolation and identification of bone marrow mesenchymal stem cells: After euthanasia, mice were immersed in 75% ethanol, and the tibia and femur, including both ends of the tibia and femur epithelium, were extracted under sterile conditions. After flushing the bone marrow cavity with PBS, the cell suspension was blown away to allow the cells to flow out, and this was repeated three times. The bone marrow cell suspension was centrifuged at 1000r / min for 5min, the supernatant was removed, and the cell pellets were resuspended. This process was repeated three times, and the cell suspension was mixed with low-glucose DMEM (China Purella) containing 15% FBS and inoculated into a cell culture bottle. The culture environment was a sterile environment at 37°C, which is conducive to epiphytic growth.

[0040] Extraction of BMSC-derived exosomes: BMSCs-derived exosomes (EXO) were isolated from FBS-free BMSCs supernatant by differential centrifugation using a previously described protocol. The supernatant was centrifuged at 300 × g for 10 min, 2000 × g for 10 min, and 10,000 × g for 30 min at 4°C to remove cell debris, the pellet at the bottom of the tube was discarded, the supernatant was collected, and exosomes were extracted by centrifugation at 100,000 × g for 70 min. The exosome microspheres were washed three times with sterile PBS and stored at -80°C for later use.

[0041] Synthesis of PDA@CQ: Preparation of PDANPs using a typical self-polymerization method Briefly, 18 mg of dopamine was fully dissolved in 9 mL of deionized water and polymerized under alkaline conditions (84 μL of 1 M NaOH) at 60 °C for 5 h. After centrifugation and three washes with MeOH, the purified PDANPs were dispersed in MeOH, and 5 mg of CQ was added to the suspension. After stirring at ambient conditions for 24 h, PDA@CQ NPs were collected by centrifugation and subsequent vacuum drying. The morphology of PDA NPss and PDA@CQ NPs was evaluated by transmission electron microscopy (TEM), and the DPPH, ABTS, and PTIO free radical scavenging abilities of PDANPs were tested by UV-visible spectroscopy (UV-Vis).

[0042] Ultrasonication: siFDX1 or siRNA-Cy3 was loaded onto EXO using an ultrasonication system from Sonic & Materials, Inc. (Newtown, USA). EXO and siFDX1 (Table S1) were mixed in PBS at a 1:1 (mass / mass) ratio to give a final EXO concentration of 4 μg / mL. The ultrasonication settings used were 20% amplitude, six 30-second on / off cycles, with a 2-minute cooling period between each cycle. After ultrasonication, the unbound siFDX1 in the supernatant was assessed at 260 nm using a microplate reader. The amount of siFDX1 in the supernatant was determined by the weight ratio of encapsulated siFDX1 to loaded siFDX1 nanovesicles. The solution was then incubated at 37°C for 60 min to restore the exosome membrane and then stored at 4°C for later use when possible. To confirm that siFDX1 was successfully encapsulated by the exosome membrane, the exosomes were stained with Dio and loaded with siFDX1-Cy3.3, and after ultrasonic incubation, the colocalization of Cy3 and Dio was observed under a fluorescence microscope.

[0043] Nano-contact vortex technique: PBS was added to EXOsiFDX1 exosomes to prepare a suspension, which was sterilized by filtration through a 0.22 μm filter membrane. Then, PDA-CQ solution (0.5 mg / mL) was prepared with distilled water and added to the EXO-containing exosomes at a ratio of 2:1 (mass / mass). siFDX1 The mixture of extracellular vesicles was vortexed to prepare EXO siFDX1-PDA@CQ The suspension was incubated at 37°C for 24 h, then ultracentrifuged at 50,000 x g for 70 min to remove free PDA-CQ particles, and the isolated exosomes were stored at -20°C for further analysis. siFDX1-PDA@CQ Dynamic light scattering (DLS, Malvern Panalytical, UK) was used to determine the particle size and zeta potential of the exosomes, and ultraviolet-visible spectrophotometer (UV-Vis) was used to verify the loading of CQ on the exosomes.

[0044] EXO siFDX1-PDA@CQIn vitro function of the complex: A copper death cell model was prepared using Eles (MedChemExpress, USA) and CuCl2 (Cu, SigmaAldrich, USA). The experimental groups included the control group, Eles+Cu, Eles+Cu+EXO, Eles+Cu+EXOsiFDX1, and Eles+Cu+EXOsiFDX1-PDA@CQ groups. Cell proliferation and migration abilities were measured, and the intracellular Cu+ level was evaluated using the Cu ion probe CS-1 (MedChemExpress, USA). The results were observed and photographed using a fluorescence microscope. When the confluence of NIH-3T3 and HUVECs reached 30%–50%, EXO was added. siFDX1-PDA@CQ (5 μg / well) complexes, and after incubation, images were randomly taken and observed.

[0045] The copper-induced cell death model was prepared using H2O2 (SigmaAldrich, USA). The experimental groups included control group, H2O2, H2O2+EXO, H2O2+EXO siFDX1 and H2O2+EXO siFDX1-PDA@CQ Groups were used to measure cell migration and tube formation abilities.

[0046] qRT-PCR: Total RNA was isolated using the Ultrapure RNA Kit (Yeasen, China) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using RT kit (Yeasen, China), and then qRT-PCR was performed on StepOne platform (Yeasen, China).

[0047] Preparation and evaluation of hydrogels: The gel precursors of OSA and PGA-g-ADH were synthesized by chemically modifying natural biomacromolecules. Typically, 1 g of sodium alginate was dissolved in 100 mL of deionized water, and then 0.6 g of NaIO4 was added to the solution and stirred in the dark for 24 h. The mixture was dialyzed with deionized water for three days, and the OSA product was finally obtained by freeze drying. Then, 2 g of PGA (MW = 700 kDa) was dissolved in 200 mL of methyl methanesulfonate buffer (pH = 6.5). Then, 4 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.8 g of N-hydroxysuccinimide were added to the PGA solution and stirred for 30 min. Then, 9 g of ADH was added to the mixed solution and stirred for another 24 h. The reaction product was dialyzed with deionized water for three days and then freeze dried to obtain PGA-g-ADH. The prepared OSA and PGA-g-ADH were verified by 1H NMR, FTIR, GPC and Zeta potential experiments. A series of hydrogels were prepared by mixing the two precursors. Generally, EXO siFDX1-PDA@CQ(2 μg / ml) was dissolved in a 6 wt% solution of OSA- to obtain solution A. After mixing 6 wt% PGA-g-ADH and CP 05 polypeptide (CP05-Ep) (2 μg / ml), the hydrogel spontaneously gelled when the precursor solutions A and B were mixed at equal volumes. The gelation time of the customized hydrogels was determined by the vial inversion method. The dynamic rheological properties of various hydrogel samples were measured using a rheometer. In order to determine the strain-responsive rheological behavior, the frequency was fixed at 0.1 Hz in the strain range from 0.1% to 100%. The gel strength was calculated based on the strain sweep test performed previously. The frequency-responsive rheological properties were tested in the frequency range of 0.1-10 Hz at 1% strain. The shear-thinning behavior of the hydrogel was verified at a shear rate of 0.1 to 100 Hz in the viscosity mode. The temperature of the entire rheological measurement process was set to 37 °C. The ROS scavenging performance of the hydrogel was studied using the above method. The porous structure of the freeze-dried hydrogel was observed by scanning electron microscopy. Hemolysis test was used to evaluate the blood compatibility of customized hydrogels. Whole blood was collected from healthy pigs. Afterwards, different hydrogels with different concentrations were incubated with 4% diluted blood at 37°C for 120 min. The hemolysis test was performed using a microplate reader (MD Pro5) The absorbance of the supernatant was measured at 540 nm to quantify the rate of hemolysis. Physiological saline and Triton X-100 were used as negative and positive controls, respectively.

[0048] Animal experiments: All animal experiments were approved by the Experimental Animal Ethics Committee of the Second Hospital of Zhejiang University (No. 2024-137). For animal experiments, six-week-old male C57BL / 6 mice were intraperitoneally injected with STZ 50 mg / kg for five consecutive days. One week later, blood glucose was measured using a blood glucose monitor. Blood glucose levels above 16.7 mM indicated successful induction of diabetes. The mice were anesthetized with 1% (50 mg / kg) sodium pentobarbital (Sigma-Aldrich), and then the back hair was removed. After disinfection, a circular full-thickness skin wound with a diameter of 8 mm was formed on the back skin. Pressure applied to the wound effectively stopped bleeding and further reduced bleeding. Subsequently, a silicone ring was fixed to the back skin of the mice using an adhesive to prevent skin contraction. The mice were divided into a control group (PBS), an OP group (gel only), an OP+EXO group (simple exosomes), and an OP+EXO group (simple exosomes). siFDX1 Group (4 μg / wound, exosomes carrying siFDX1) and OP+EXO siFDX1-PDA@CQGroup (4 μg / wound, exosomes carrying siFDX1 and PDA@CQ), finally, the wound was covered with vaseline gauze (3M, Tegaderm, USA) and transparent dressing, and the dressing was changed on postoperative days 3 and 6. Photos were taken on postoperative days 0, 3, 7, and 14, and the wound area was measured using ImageJ software (NIH, ImageJ 1.8, USA).

[0049] Histological analysis: On the 7th and 14th days after injury, the entire injury bed of each group of mice was subjected to histological analysis. Skin tissue samples from diabetic mice and normal mice were stored at room temperature and histological and Western blotting analyses were performed at -80°C. The wound tissue was fixed with 4% glutaraldehyde. After dehydration, the tissue was embedded in paraffin and cut into 8 μm thick longitudinal sections for staining. HE staining was used to observe the re-epithelialization rate of the wound tissue, and Masson staining was used to observe the accumulation of collagen. In addition, immunofluorescence was used to observe the expression of FDX1.

[0050] Small animal in vivo imaging: As described above, a circular full-thickness skin wound with a diameter of 8 mm was established, and the mice were divided into an injection group (injected with exosomes with PKH26 fluorescence) and an OP group (hydrogel encapsulated with exosomes with PKH26 fluorescence). Finally, the wound was covered with vaseline gauze (3M, Tegarderm, USA) and a transparent dressing. The in vivo imaging device was used to take pictures on days 0, 1, 2, 3, and 5 after surgery, and the fluorescence retention of the wound was recorded.

[0051] Pig full-thickness wound healing model: Panamanian mini pigs weighing 20-25 kg were used in this example. Approved by the Animal Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine (No. 2024-138), after general anesthesia, the back hair of the mini pigs was removed, the back was disinfected with iodine and 70% ethanol, and 6 circular full-thickness skin wounds (2 cm in diameter, 3 on each side) were created and divided into three groups, with a distance of 4 cm between each group. After surgery, the pigs were randomly assigned to receive PBS (control group), OP (hydrogel only), and OP+ (EXO siFDX1-PDA@CQ All wounds were immediately covered with transparent dressings (3M, Tegarderm, USA) and complete dressing changes were performed on days 7, 14, and 21, consistent with the initial administration of the same treatment drugs. Images were taken on days 0, 7, 14, 21, and 28 after surgery and analyzed using ImageJ software (NIH, ImageJ 1.8, USA).

[0052] Statistical analysis: Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0 software (La Jolla, CA, USA). Student's t test was used to evaluate the differences between the two groups. One-way ANOVA with Tukey's multiple comparison test was used to compare multiple groups. Statistical significance was defined as *P < 0.05 or **P < 0.01. ns indicated no statistical significance.

[0053] Cuproptosis is caused by intracellular copper ion (Cu) overload and overexpression of ferredoxin 1 (FDX1), which has been identified as a regulator of the skin wound healing process. This example demonstrates the presence of cuproptosis in the skin wound bed and reactive oxygen species-induced cell models. In order to address the two pathways leading to cellular cuproptosis, a nanomedicine-engineered exosome is proposed. A Cu-chelator (Clioquinol, CQ) polydopamine (PDA)-modified stem cell exosome containing siRNA-FDX1 is designed, named EXOsiFDX1-PDA@CQ, which can effectively inhibit the two pathways of cuproptosis. The functionalized exosomes are loaded into an injectable gel for the treatment of diabetic wounds in mice and acute skin wounds in pigs. The localized and controlled release of EXOsiFDX1-PD@CQ ensures the retention of the therapeutic agent on the wound bed, effectively promoting wound healing. The engineered exosome strategy with functional nanoparticles (NPs) proposed in this example provides an effective and scalable new approach to regulate cuproptosis.

[0054] Example: Nanomedicine-modified engineered exosomes (EXO siFDX1-PDA@CQ ) Therapeutic Application Experiment

[0055] Exosomes have recently been widely used as gene carriers due to their unique lipid bilayer structure, excellent biocompatibility and easy accessibility. These vesicles have shown promising therapeutic effects in many animal disease models and clinical studies. Moreover, due to the different loading efficiency and molecular stability of miRNA, siRNA and drugs in liposomes, liposomes are currently mainly used as single substance delivery carriers. Recently, the modification of catechol nanomedicines has become one of the most effective methods for upgrading exosomes. Exosomes are widely used to cover various surfaces, such as bacteria and cells, to achieve controlled drug delivery. Exosomes are usually applied to the wound bed by traditional periwound injection methods, which can lead to uneven drug distribution and rapid loss of drugs within a few hours, thus affecting the therapeutic effect. This often requires repeated injections, causing inconvenience and additional treatment costs. When exosomes are encapsulated in injectable hydrogels, uniform drug distribution can be achieved on the wound bed, while reducing drug dosage and promoting slow and controlled release. Therefore, the exosome-anchored peptide CP05-enhanced injectable hydrogel enables longer drug release.

[0056] like Figure 1 The figure shows the engineered exosomes modified with nanomedicine (EXO siFDX1-PDA@CQ ) Schematic diagram of the workflow of therapeutic applications, EXO siFDX1-PDA@CQ Decomposition of PDA particles by endocytosis releases drug-chelated copper and releases siRNA by exosome rupture, EXO siFDX1-PDA@CQ Encapsulated in injectable exosomes with sustained-release hydrogels, the exosomes showed a strong effect in promoting wound healing by reducing intracellular copper content and reducing FDX1 expression, and by synergistically inhibiting copper death; the existence of FDX1-mediated copper death in skin wounds and ROS-induced cell models was confirmed by colorimetry, RNA sequencing (RNA-seq), and immunofluorescence experiments ( Figure 1 A), then, through the specially designed engineered exosomal polydopamine (PDA)@clioquinol (CQ) (EXO siFDX1-PDA@CQ ) is aimed at Figure 1 The dual-pathway mechanism of copper death shown in B. In short, CQ is an effective copper ion chelator that can be conveniently loaded into PDA nanoparticles (NPs). A PDA@CQ nanomedicine was developed. PDA@CQ NPs were loaded onto the outer membrane of engineered exosomes through surface adhesion, covalent and non-covalent binding interactions. The siRNA-FDX1 in the engineered exosomes achieved a strong inhibition of FDX1 expression, solving the problem of a single gene regulation mode in exosome delivery. In order to obtain a longer drug retention time, the CP05-enhanced injectable hydrogel was used as a transport vehicle to administer EXO. siFDX1-PDA@CQ ,like Figure 1 As shown in C, the hydrogel EXO siFDX1-PDA@CQ injected into the wound bed of diabetic mice and rats. Based on these results, EXO siFDX1-PDA@CQ A matched dual-pathway approach enhances copper's death-inhibiting effect, thereby promoting wound healing.

[0057] The prior art emphasizes that oxidative stress exists in skin wounds and stimulates the increase of intracellular copper. Excessive copper in cells leads to increased expression of iron-sulfur cluster proteins, resulting in copper death. In order to verify the experimental results of the above skin wounds, the total copper concentration in the skin wound tissue was detected by colorimetry, such as Figure 2The figure shows that copper death hinders the healing of diabetic wounds, including (A) copper content in healthy mice and diabetic mice, (BC) detection of FDX1 expression levels in wound groups of healthy mice and diabetic mice, FDX1 (green); DAPI (blue), scale = 100mm (left) or 20μm (right), (D) PCA and DEGs analysis results of diabetic and normal wounds, (E) enrichment analysis of 867 DEGs, (F) FDX1 mRNA expression in volcano plot, (GH) Western blot was used to detect the expression of copper death proteins in wounds of healthy mice and diabetic mice at different stages, and the results were analyzed, (IL) Western blot was used to detect the expression of copper death-related proteins in two types of wound cells under different H2O2 stimulations, and the results were analyzed. Data are expressed as mean ± SD. *P<0.05, **P<0.01, all data represent at least three independent experiments, and the copper content in diabetic wounds is significantly higher than that in healthy wounds ( Figure 2 A).

[0058] Meanwhile, the prior art also states that the occurrence of copper overload is related to copper overload and is also closely related to the upregulation of FDX1. There is no report showing that there is a correlation between oxidative stress in diabetic wounds and copper death caused by copper overload, and the specific role of FDX1 in diabetic wounds is unclear. Therefore, this example evaluates the presence of FDX1 protein in diabetic mice by immunofluorescence, such as Figure 2 As shown in BC, the FDX1 protein level in diabetic wounds was significantly higher than that in healthy mice (P<0.01). To further reveal the pathophysiological mechanism of diabetic wounds, RNA-seq analysis was performed on diabetic and normal wounds. The results of principal component analysis (PCA) showed that there were significant differences in gene expression between the two groups ( Figure 2 D), differentially expressed genes (DEG) analysis showed that compared with the normal group, there were 508 down-regulated genes and 359 up-regulated genes in the diabetic group ( Figure 2 E), these downregulated genes are related to skin, cartilage and cartilage development, indicating that the wound repair process in diabetic mice is impaired, which is consistent with the immunofluorescence results of this example. FDX1 is a key regulator of copper death and is significantly upregulated in diabetic wounds (P<0.05) ( Figure 2 F).

[0059] Ovarian development-related protein lipoyl-dihydrolipoamide S-acetyltransferase (Lip-DLAT) and FDX1 were subsequently detected in wound samples of healthy and diabetic mice. The results were evaluated by grayscale intensity analysis, which showed that healthy mouse wounds rarely expressed proteins related to oocyte production. However, as the duration of diabetes increased, the expression of ovarian development-related proteins Lip-DLAT and FDX1 increased significantly (P<0.05), indicating that more cells were undergoing ovarian development ( Figure 2 GH). Fibroblasts (NIH-3T3) and human umbilical vein endothelial cells (HUVECs) were used to establish a cell model in vitro. Different concentrations of hydrogen peroxide (H2O2) were used to simulate the oxidative stress of diabetic wounds at different stages. Ovarian development-related proteins were detected by Western blotting. The results showed that the expression level of ovarian development-related proteins increased synchronously with the increase of H2O2 concentration ( Figure 2 IL), in addition, this example detected the expression level of FDX1 in the two cells under different glucose concentrations. As the glucose concentration increased, the expression level of the gene also increased, and there was a statistical difference with the control group (P<0.05). These findings indicate that both excessive ROS and high glucose levels contribute to the gradual increase in the expression level of proteins related to oocyte production.

[0060] In the construction of engineered exosomes in this embodiment, the wound cell death caused by oocyte apoptosis is effectively reduced, such as Figure 3 As shown, the engineered exosomes modified by nanomedicine were constructed to (A) produce EXO siFDX1-PDA@CQ Workflow, (B) Morphology of PDA and PDA@CQ determined by TEM, scale bar = 50 nm, (C) Contents of different elements in PDA@CQ determined by TEM and energy dispersive spectroscopy, scale bar = 300 nm, (D) Images of three differently modified exosomes captured by TEM (black arrows: exosomes; red arrows: PDA@CQ), scale bar = 50 nm, (E) Measurement of EXO, EXO siFDX1 and EXO siFDX1-PDA@CQ The surface Zeta potential of the group, (F) detection of BMSCs, EXO, EXO siFDX1 and EXO siFDX1-PDA@CQ Surface labeling, (G) particle size measured by DLS, (H) fluorescence microscopy image showing the visual effect of siFDX1-Cy3 (red) encapsulated in Dio-stained exosomes (green), scale bar = 1 μm, (I) fluorescence microscopy image showing PKH26-stained EXO siFDX1-PDA@CQ(Red) Visual effect of successful internalization by two wound cells, scale bar = 20 μm, data are expressed as mean ± SD, *P < 0.05, **P < 0.01, all data represent at least three independent experiments; Exosomes were obtained from bone marrow mesenchymal stem cells (BMSCs) by gradient centrifugation, and then siRNA was internally loaded by typical water bath sonication technique ( Figure 3 A), a US Food and Drug Administration-approved hydrophobic copper chelator (CQ) was used and synthesized via a PDA-based nanocarrier (PDA@CQ) via a catechol self-polymerization and drug uptake method for drug encapsulation and exosome loading, and then, a nano-contact vortex shaker and modular co-culture engineering were used to develop nano-drug exosomes with gene knockout capabilities (hence “EXO siFDX1-PDA@CQ ”), after being taken up by cells, these EXO siFDX1-PDA@CQ The drug will first be released from the inside of the PDA shell after it ruptures; subsequently, the exosome membrane will decompose, and the siRNA in the exosome can be released and downregulate the expression of FDX1.

[0061] like Figure 3 As shown in Figure B, PDANPs are spherical with a diameter of 120 nm. After CQ is encapsulated into PDA to form PDA@CQ, its particle size does not change significantly. Subsequently, energy dispersive spectroscopy confirms the presence of I and Cl elements in PDA@CQ NPs, which are unique to CQ, proving that CQ is successfully encapsulated in PDANPs ( Figure 3 C), the typical loading amount of CQ in PDA@CQ is calculated to be 1.2%, as Figure 3 As shown in D, images were obtained by transmission electron microscopy (TEM). The morphology of siRNA-loaded EXO maintained a biconcave disk shape, and there was no significant change in particle size or membrane integrity compared with pure EXO. From the TEM image, the combination of EXO and PDA@CQ can be clearly observed, and the particle size increased to 200 nm.

[0062] This example also tested the particle size, Zeta potential and expression of surface markers of these nanoparticles. The results showed that due to the presence of negatively charged PDA on the surface, EXO siFDX1-PDA@CQ The membrane surface potential dropped from -27mV to -34mV ( Figure 3 E), however, with EXO or EXOs iFDX1-PDA@CQ In comparison, EXO siFDX1-PDA@CQ There were no differences in surface markers ( Figure 3 F), In addition, DLS detection showed that EXO siFDX1-PDA@CQ The particle size increased to 229.8 nm, which is consistent with the TEM results ( Figure 3 G).

[0063] To confirm that siFDX1 was successfully integrated into EXO siFDX1-PDA@CQ In the , Cy3-modified siRNA displayed red fluorescence and Dio-stained exosomes were evaluated for localization using fluorescence microscopy, as Figure 3 As shown in H, significant colocalization of red and green fluorescence was observed under a fluorescence microscope, indicating that siRNA was successfully encapsulated in EXO siFDX1-PDA@CQ In order to confirm EXO siFDX1 -PDA@CQ To be absorbed by cells, they were labeled with PKH26 with red fluorescence and incubated with cells for 24 hours. Then, the endocytosis of the cytoskeleton and nucleus was observed by fluorescence microscopy. Red fluorescent exosomes were clearly observed within the green fluorescent cytoskeleton, indicating that the cells successfully internalized EXO siFDX1-PDA@CQ ( Figure 3 I).

[0064] In the package EXO siFDX1-PDA@CQ In the construction of hydrogels, PDANPs are a well-studied nanomaterial with excellent antioxidant properties. In this example, the ROS scavenging performance of the prepared PDA@CQ NPs was evaluated in detail using semi-quantitative spectral analysis. Figure 4 A, three representative groups, 2,2-diphenyl-1-benzimidazole (DPPH), 2,2-diamino-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), can be eliminated within 30 minutes of incubation with PDA@CQ NPs. The ROS clearance rate is positively correlated with the concentration of PDA@CQ NPs. The concentration thresholds for 80% ROS clearance are 50 μg / mL and 200 μg / mL for DPPH and ABTS groups, respectively, and 200 μg / mL for PTIO, indicating that PDA@CQ NPs have effective anti-ROS properties. In summary, the prepared PDA@CQ nanoparticles have antioxidant and potential copper ion chelating abilities and can be used as effective participants in exosome surface engineering.

[0065] Subsequently, two biomacromolecules, oxidized sodium alginate (OSA) and polyglutamic acid grafted adipic acid dihydrazide (PGA-g-ADH), were synthesized. Figure 4Shown are the preparation and evaluation of hydrogels, (A) scavenging ability of PDA@CQ NPs for DPPH, ABTS, and PTIO, (B) schematic diagram of the network formation of PDA@CQ and OP hydrogels, and (C) representative images of the precursor and hydrogel preparation process. Scale bar = 1 cm, (D) Gelation time of OP hydrogels with different concentrations, (E) Gelation time of OP hydrogels with different concentrations, (F) Evaluation of frequency-responsive rheological properties of tailored hydrogels at 1% strain at 37°C, (G) Injectability of OP hydrogels, Scale bar = 1 cm, (H) Representative SEM images of OP hydrogels with different concentrations, Scale bar = 5 μm, (I) Evaluation of frequency-responsive rheological properties of tailored hydrogels at 1% strain at 37°C, (J) Analysis of frequency-responsive rheological behavior of tailored hydrogels at 37°C and gel strength of different hydrogels; An injectable hydrogel (OP) encapsulating EXOsiFDX1-PDA@CQ was prepared using Schiff base “click” chemistry of OSA, PGA-g-ADH, and thiolated vesicle anchoring ligand (CP05-Pep) to prolong the life of EXO. siFDX1-PDA@CQ Retention time in diabetic ulcers ( Figure 4 B), the synthesized OSA polymer contains aldehyde groups and rapidly gels with PGA-g-ADH and CP05-Pep at room temperature ( Figure 4 C), the gelation time of the customized hydrogel was tested using the vial inversion method. The gelation speed was positively correlated with the concentration of the two macromolecules. When the concentration of OSA and PGA-g-ADH was set to 1wt%, we were unable to capture the solidification process, but when the concentration increased to 2wt% and 3wt%, the gelation process was significantly accelerated ( Figure 4 D). In addition, the addition of CP05 Pep and PDANPs had little effect on the gelation time, highlighting the main contribution of the Schiff base reaction between OSA and PGA-g-AD H in the curing process ( Figure 4 E), the resulting OP-3wt% hydrogel stably solidified within 50 seconds without visible leakage. Considering the ease of use, 3wt% was selected as the optimal concentration for subsequent studies. To detect the distribution of EXOsiFDX1-PDA@CQ in the hydrogel, they were labeled with PKH26 red fluorescence. PKH26-labeled exosomes were evenly distributed in the hydrogel, indicating successful encapsulation of exosomes. In addition, with the increase in shear rate, the viscosity of the hydrogel decreased significantly, indicating a typical shear-thinning behavior, which facilitates injection ( Figure 4 F), the modular in situ gelation behavior of the two solutions endows the hydrogels with excellent injectability and convenient extrusion from a needle.

[0066] The blood compatibility of the obtained OP hydrogel was evaluated before in vivo administration. At the same time, the anti-hemolytic properties of the hydrogels prepared at different concentrations were studied, with Triton X-100 as a positive control to completely hemolyze and dilute the blood. Therefore, the hemolysis rates of the hydrogels at different concentrations were calculated to be less than 0.5%, indicating that the obtained hydrogels have excellent blood compatibility.

[0067] like Figure 5 As shown, EXO siFDX1-PDA@CQ Pathways synergistically inhibit copper death in time and space, (A) EXO siFDX1-PDA@CQ Schematic diagram of the mechanism of synergistic inhibition of copper death. (BE) CCK8 was used to detect the occurrence of copper death in cells after siRNA knockdown, and morphological changes were observed under an optical microscope. Scale bar = 200 μm. (F) EXO siFDX1-PDA@CQ The degree of cell survival improvement after treatment, (G) qRT-PCR detection of EXO siFDX1-PDA@CQ Down-regulation of FDX1 in cells after treatment. (HI) Fluorescence microscopy to detect intracellular copper ion concentration after different treatments. Scale bar = 100 μm. (JK) Western blot to detect EXO siFDX1-PDA@CQ Expression of copper cell apoptosis-related proteins in cells after treatment and statistical analysis were performed. (LM) Transwell assay was used to detect the migration ability of cells in each group. Scale bar = 100 μm. Data are expressed as mean ± SD, *P < 0.05, **P < 0.01. All data were verified by at least 3 independent experiments; siFDX1-PDA@CQ In the synergistic inhibition of extrusion, EXO siFDX1-PDA@CQ It is possible that the inhibition of oocyte production through two pathways simultaneously can achieve dual synergistic effects at the spatiotemporal and molecular levels ( Figure 5 A), then, two different cell type ovariogenesis models were constructed using Elesclomol (Eles, a Cu2+ ionomer known for its role in ovariogenesis) and copper chloride (CuCl2), and these oocyte generation models showed that FDX1 downregulation significantly inhibited cell death (P<0.05) ( Figure 5 B and D), while maintaining normal cell morphology ( Figure 5 C and E), general verification of cell viability using CCK8 and cell viability staining to validate EXO siFDX1-PDA@CQ The results show that EXO siFDX1 and EXO siFDX1-PDA@CQ Both can effectively inhibit cell death, among which EXO siFDX1-PDA@CQ Show more than EXO siFDX Better effect (P<0.05)( Figure 5 F). Intracellular FDX1 levels increased after the addition of Eles and CuCl2.siFDX1 and EXO siFDX1-PDA@CQ Treatment effectively reduced the expression of FDX1 mRNA (P>0.05)( Figure 5 G), indicating that PDA@CQ on the surface of exosomes does not affect the function of internal siRNA. In contrast, the CS-1 probe was used to detect intracellular Cu + Statistical analysis of their fluorescence intensities showed that downregulation of FDX1 could partially inhibit the overload of Cu 2+ Converted to Cu + , but the addition of PDA@CQ significantly enhanced this effect (P<0.05) ( Figure 5 HI), and then, Western blotting and qRT-PCR results confirmed the successful downregulation of FDX1 expression level. These results indicate that EXO siFDX1 and EXO siFDX1-PDA@CQ can effectively downregulate FDX1. In addition, EXO siFDX1-PDA@CQ Q's Lip-DLAT downregulation effect is better than EXO's siFDX1 (P<0.05)( Figure 5 JK), In addition, to evaluate whether follicle growth inhibition can restore cell migration ability, we performed cell transwell assays. Based on our results, EXO siFDX1-PDA@CQ The cells in the group showed the best cell migration ability.

[0068] In addition, this embodiment also detects that EXO siFDX1-PDA@CQ We found that EXO siFDX1-PDA@CQ The migration ability of cells can be restored and compared with H2O2 group and H2O2+EXO siFDX1 Compared with the EXO siFDX1-PDA@CQ The scratch area of ​​cells in the group was the smallest, and the most significant difference was observed at 48 hours (P < 0.05) (Figure S13). The tube formation experiment of HUVECs also showed that EXO siFDX1-PDA@CQ The tube-forming ability of cells can be restored. In addition, qRT-PCR results showed that EXO siFDX1-PDA@CQ After treatment, the mRNA levels of VEGFA (vascular endothelial growth factor A) in HUVECs and Col-I (collagen I) in NIH-3T3 cells were the highest (P<0.05).

[0069] like Figure 6 As shown, EXO siFDX1-PDA@CQEffectively promotes diabetic wound healing, (A) Schematic diagram illustrating the establishment and treatment of diabetic wound mouse model (created by BioRender.com), (B) Representative images of diabetic wounds in each group during the healing process, (C) Simulated diagram of wound closure area, (D) Statistical analysis of wound healing rates in different groups on day 7, (E) Statistics of wound healing rates within 0-14 days, (FG) In vivo imaging was used to determine the PKH26-labeled EXO after injection and OP hydrogel administration siFDX1-PDA@CQ Survival time and content, (H) Copper content in tissues was detected by colorimetric method, data are expressed as mean ± SD, *P < 0.05, **P < 0.01, all data represent at least three independent experiments; in EXO siFDX1-PDA@CQ It can promote wound healing. In order to evaluate EXO siFDX1-PDA@CQ In vivo efficacy, streptozotocin (STZ) was injected intraperitoneally to establish a typical type I diabetic mouse model. Continuous readings of more than 16.7mmol / L indicated that the diabetes model had been successfully established. In addition, the body weight and blood glucose levels of the mice were monitored regularly. The results showed that the body weight decreased over time, although there was no statistically significant difference between the groups. Subsequently, a circular full-thickness skin wound with a diameter of 8 mm was created on the back of the mice ( Figure 6 A). ImageJ software (NIH, ImageJ 1.8, USA) was used to analyze the wound closure rate. Figure 6 As shown in B, on the 14th day, OP+EXO siFDX1-PDA@CQ The wound area of ​​the group healed and was covered by new epithelial cells, while the other four groups still showed open wounds and scabs. The results were statistically analyzed and Figure 6 As can be clearly seen in the wound area graph of C, no significant difference in healing rate was observed among the PBS, OP, and OP+EXO groups on day 7. siFDX1 The healing rate of the OP+EXO group was significantly higher than that of the PBS, OP and OP+EXO groups (P<0.01). siFDX1-PDA@CQ group showed the highest healing rate (P<0.05) ( Figure 6 D) In ​​addition, according to the analysis of wound healing rates from 0 to 14, EXO siFDX1-PDA@CQ The group showed the best effect among the five groups ( Figure 6 E) Injection and incorporation of EXO siFDX1-PDA@CQ A comparative analysis of the retention rates of OP capsules was conducted to verify the drug retention rate using the IVIS Lumina series. The results showed that OP was significantly superior to EXO siFDX1-PDA@CQ The sustained-release effect of the injection group was better than that of the injection group, and the effect lasted for up to 3 days. On the 5th day, the content of exosomes on the wound surface was significantly reduced. Therefore, we changed the dressing on the 3rd and 6th days ( Figure 6FG), in addition, the colorimetric method was also used to detect the concentration of Cu in wound tissue. siFDX1-PDA@CQ Administration of medication could reduce Cu levels (P<0.01) ( Figure 6 H). Figure 7 As shown in AD, OP+EXO siFDX1 The wound length of the OP+EXO group was significantly shorter than that of the PBS, OP, and OP+EXO groups (P<0.01). siFDX1-PDA@CQ The group performed better than OP+EXO siFDX1 The wound healing rate was higher in the group (P<0.05). Figure 7 EF) can be seen that OP+EXO siFDX1-PDA@CQ Group comparison: PBS, OP, OP+EXO and OP+EXO siFDX1 The group showed significantly increased collagen deposition and a more organized pattern of fibroblasts. In summary, EXO siFDX1-PDA@CQ Effectively promote the healing of diabetic wounds.

[0070] like Figure 7 As shown, EXO siFDX1-PDA@CQ Accelerate the healing of diabetic wounds by reducing Cu content and inhibiting the expression of FDX1. (AD) H&E staining images of wound samples in different groups were quantitatively analyzed at 7 days and 14 days, and the wound length was quantitatively analyzed. Scale bar = 1mm (left) or 100μm (right). (EF) Masson staining images and collagen content statistics of wound samples in different groups on the 14th day after surgery. Scale bar = 1mm (left) or 100μm (right). (G) FDX1 mRNA levels in different stem cell groups were determined by qRT-PCR. (HI) Western dot blot analysis of Lip-DLAT and FDX1 expression levels. (JK) FDX1 expression images of tissues in different groups under fluorescence microscopy, FDX1 (green); DAPI (blue), scale bar = 100μm. Data are expressed as mean ± standard deviation, *P<0.05, **P<0.01, all data represent at least three independent experiments; to further validate EXO siFDX1-PDA@CQ In vivo, the FDX1 mRNA level was assessed using qRT-PCR ( Figure 7 G), the results show that EXO siFDX1 and EXO siFDX1-PDA@CQ The mRNA level of FDX1 was successfully downregulated (P<0.01). In addition, Western blot results showed that the expression of FDX1 and Lip-DLAT was significantly reduced ( Figure 7 HI), and immunofluorescence experiments also proved that EXO siFDX 1 group and EXO siFDX1 -PDA@CQThe expression of FDX1 in the Figure 7 JK), EXO siFDX1-PDA@CQ The expression level of Lip-DLAT in the EXO group was significantly reduced, indicating that copper death was significantly inhibited. These findings are consistent with the results of in vitro experiments. In addition, hematoxylin and eosin (H&E) staining was performed on key organs of each group, including the heart, liver, spleen, lungs, and kidneys. The results showed that both the hydrogel and EXO siFDX1-PDA@CQ None of them caused obvious damage to these organs, indicating good tissue biocompatibility.

[0071] like Figure 8 As shown, functional validation of engineered exosomes modified with nanomedicine in a full-thickness porcine wound model, (A) Schematic diagram showing the establishment and treatment of the full-thickness porcine wound model (created by BioRender.com), (B) Representative images of the wound healing process in each group. (C) Quantitative evaluation of wound closure rate on day 21 after surgery, (D) Detection of FDX1 mRNA expression levels in healthy skin and wound tissues in each group, (EF) H&E staining images of wound samples in different groups on day 21 after surgery, quantitative analysis of wound length, scale bar = 5 mm (left) or 0.5 mm (right), (GH) Masson staining images of wound samples in each group on day 21 after surgery, quantitative analysis of wound collagen deposition, scale bar = 5 mm (left) or 0.5 mm (right), data are expressed as mean ± SD, *P < 0.05, **P < 0.01, all data represent at least three independent experiments; in EXO siFDX1-PDA@CQ In the wound healing model that can promote full-thickness pig skin wounds, pigs and humans have similar wound healing processes. We will siFDX1 -PDA@CQ The effectiveness of the study was extended to a full-thickness porcine wound model ( Figure 8 A), Compared with small wounds, repairing large skin defects requires longer granulation tissue formation and maturation time to cover the wound, which involves multiple growth factors and cell interactions. During the wound healing process, any disorder event may lead to refractory wounds. Large wounds have immune microenvironment barriers; therefore, a full-thickness skin defect wound with a diameter of 2 cm was established on the back of the pig. Digital photos of the wounds in each group showed that compared with the PBS group, OP+EXO siFDX1-PDA@CQ The wound healing rate was the highest in the group Figure 8 B), the most significant effect was observed on day 21 (P<0.05) ( Figure 8 C). Subsequently, on the 21st day, wound and normal skin tissue samples were collected and RNA was extracted. According to the results of qRT-PCR, the expression level of FDX1 in the wound was significantly higher than that in the normal skin (P<0.01), while EXO siFDX1-PDA@CQThe administration of α-glucose significantly downregulated the expression level of FDX1 (P<0.01) ( Figure 8 D), 28 days after surgery, OP+EXO siFDX1-PDA@CQ The group showed almost completely healed wounds, in sharp contrast to the OP and PBS groups, where the wounds remained open with visible scabs, such as Figure 8 As shown in EF, on day 28, there was no significant difference in wound length between the OP and PBS groups; however, the OP+EXO siFDX1-PDA@CQ The wound length of the EXO group was significantly shorter than that of the OP and PBS groups, and the wound was almost completely re-epithelialized (P<0.05). At the same time, statistical analysis showed that there was no significant difference in collagen deposition between the OP and PBS groups on day 28, and the OP+EXO group had a significantly shorter wound length than the OP and PBS groups, and the wound was almost completely re-epithelialized (P<0.05). siFDX1-PDA@CQ The collagen deposition level in the OP and PBS groups was significantly higher (P<0.05). Figure 8 GH), the experimental results confirmed that EXO siFDX1-PDA@CQ It exhibits excellent effects and superior quality in promoting wound healing, highlighting its potential therapeutic potential in clinical applications.

[0072] Experimental conclusion:

[0073] In summary, the results in this example show that copper death exists in refractory wounds, and its mediated pathways include copper overload and FDX1 upregulation. siFDX1-PDA@CQ CQ can be released to chelate copper ions in cells, thereby immediately inhibiting copper death. Subsequently, internal siRNA is released to interfere with mRNA transcription and translation, preventing the long-term occurrence of copper death by downregulating FDX1 expression. This strategy effectively inhibits copper death and promotes wound healing by combining dual-pathway and dual-time nanomedicine-modified engineered exosomes. Given the presence of copper death in refractory wounds, combining these engineered exosomes with sustained-release gels may become a feasible and useful therapeutic strategy for the management of refractory wounds in the clinic.

[0074] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. An exosome that synergistically inhibits copper death based on dual pathways, characterized by: The exosomes are polydopamine-modified stem cell exosomes, and the polydopamine-modified stem cell exosomes are loaded with a copper chelator and siRNA-FDX1, and the siRNA-FDX1 is used to inhibit the expression of ferredoxin 1.

2. The method for preparing exosomes based on dual-pathway synergistic inhibition of copper death according to claim 1 is characterized in that: The following steps are involved: S1, extracting exosomes from bone marrow mesenchymal stem cells using gradient centrifugation; S2, using water bath sonication technology to load siRNA-FDX1 into exosomes; S3, preparing polydopamine nanoparticles by a self-polymerization method and loading a copper chelator to form a nanodrug; S4. The nanodrugs loaded with copper chelators such as polydopamine were combined with the surface of exosomes through nano-contact vortex technology to complete the preparation of exosomes based on dual-pathway synergistic inhibition of copper death.

3. Use of exosomes based on dual-pathway synergistic inhibition of copper death according to claim 1 in promoting wound healing.

4. Use of the exosomes based on dual-pathway synergistic inhibition of copper death according to claim 1 in the preparation of a drug for treating wound healing.

5. A pharmaceutical composition, characterized in that: The composition includes the exosomes based on dual-pathway synergistic inhibition of copper death and a hydrogel carrier as described in claim 1, wherein the hydrogel carrier encapsulates the exosomes based on dual-pathway synergistic inhibition of copper death, and is used to prolong the wound retention time of the exosomes based on dual-pathway synergistic inhibition of copper death.

Images